Runx1 contributes to neurofibromatosis type 1 neurofibroma formation.

Neurofibromatosis type 1 (NF1) patients are predisposed to neurofibromas but the driver(s) that contribute to neurofibroma formation are not fully understood. By cross comparison of microarray gene lists on human neurofibroma-initiating cells and developed neurofibroma Schwann cells (SCs) we identified RUNX1 overexpression in human neurofibroma initiation cells, suggesting RUNX1 might relate to neurofibroma formation. Immunostaining confirmed RUNX1 protein overexpression in human plexiform neurofibromas. Runx1 overexpression was confirmed in mouse Schwann cell progenitors (SCPs) and mouse neurofibromas at the messenger RNA and protein levels. Genetic inhibition of Runx1 expression by small hairpin RNA or pharmacological inhibition of Runx1 function by a Runx1/Cbfß interaction inhibitor, Ro5-3335, decreased mouse neurofibroma sphere number in vitro. Targeted genetic deletion of Runx1 in SCs and SCPs delayed mouse neurofibroma formation in vivo. Mechanistically, loss of Nf1 increased embryonic day 12.5 Runx1(+)/Blbp(+) progenitors that enable tumor formation. These results suggest that Runx1 has an important role in Nf1 neurofibroma initiation, and inhibition of RUNX1 function might provide a novel potential therapeutic treatment strategy for neurofibroma patients.

Phase I trial of pegylated interferon-alpha-2b in young patients with plexiform neurofibromas.

OBJECTIVE: Interferon has antiproliferative and antiangiogenic properties. We sought to evaluate preliminary efficacy and determine the recommended phase II dose (RP2D) for pegylated interferon-α-2b (PI) in patients with unresectable progressive or symptomatic plexiform neurofibromas (PN). METHODS: PI was administered weekly in cohorts of 3-6 patients during the dose-finding phase and continued for up to 2 years. Twelve patients were treated at the RP2D to further evaluate toxicity and activity. RESULTS: Thirty patients (median age 9.3 years, range 1.9-34.7 years) were enrolled. No dose-limiting toxicity (DLT) was seen in patients treated at the 3 µg/kg dose level (DL) during the first 4 weeks. All 5 patients treated at the 4.5 µg/kg DL came off study or required dose reductions for behavioral toxicity or fatigue. Similar DLT on the 3 µg/kg DL became apparent over time. There was 1 DLT (myoclonus) in 12 patients enrolled at the 1.0 µg/kg DL. Eleven of 16 patients with pain showed improvement and 13 of 14 patients with a palpable mass had a decrease in size. Five of 17 patients (29%) who underwent volumetric analysis had a 15%-22% decrease in volume. Three of 4 patients with documented radiographic progression prior to enrollment showed stabilization or shrinkage. CONCLUSIONS: The RP2D of PI for pediatric patients with PN is 1 µg/kg/wk. Clinical and radiographic improvement and cessation of growth can occur. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that pegylated interferon-α-2b in patients with unresectable, progressive, symptomatic, or life-threatening PNs results in radiographic reduction or stabilization of PN size.

Characteristics of children enrolled in treatment trials for NF1-related plexiform neurofibromas.

OBJECTIVE: To describe the characteristics of children enrolled in treatment trials for neurofibromatosis type 1 (NF1)-related plexiform neurofibroma (PN), PN tumor burden, PN-related complications, and treatment outcomes and to highlight the differences between characteristics of children with NF1 vs children with cancers entered on early phase drug trials. METHODS: Pre-enrollment characteristics and complications of PN were retrospectively analyzed in a cohort of 59 children with NF1-related PN treated on 1 of 7 clinical trials at the NIH between 1996 and 2007. Outcome was analyzed in a subset of 19 patients enrolled in phase I trials. Comparisons to children with cancer were made from a similar analysis performed recently. RESULTS: The median age at enrollment was 8 years. The median PN volume was 555 mL. Most patients had no prior chemotherapy or radiation, but nearly half had previous surgery for PN. PN-associated complications and NF1 manifestations were common, including pain (53%), other tumors (18%), and hypertension (8%). Investigational drug therapy was well tolerated. A median of 10 treatment cycles was administered. Patients with NF1-related PN were younger, had better performance score, had less prior therapy, and remained on study longer than cancer patients. CONCLUSIONS: Children with NF1-related plexiform neurofibroma (PN) enrolled in clinical trials had large tumors with substantial morbidity. Clinical trials in these children provide information about drug tolerance, cumulative toxicity, and pharmacokinetics in a younger population than early phase pediatric cancer trials. This report may aid in the evaluation of the applicability of traditional pediatric cancer trial designs and endpoints for NF1-related PN.

NF1 plexiform neurofibroma growth rate by volumetric MRI: relationship to age and body weight.

OBJECTIVE: To longitudinally analyze changes in plexiform neurofibroma (PN) volume in relation to age and body growth in children and young adults with neurofibromatosis type 1 and inoperable, symptomatic, or progressive PNs, using a sensitive, automated method of volumetric MRI analysis. METHODS: We included patients 25 years of age and younger with PNs entered in a natural history study or in treatment trials who had volumetric MRI over > or =16 months. RESULTS: We studied 49 patients (median age 8.3 years) with 61 PNs and a median evaluation period of 34 months (range 18 to 70). The PN growth rates varied among patients, but were constant within patients. Thirty-four patients (69%) experienced > or =20% increase in PN volume during the observation period. PN volume increased more rapidly than body weight over time (p = 0.026). Younger patients had the most rapid PN growth rate. CONCLUSIONS: Volume increase of plexiform neurofibromas is a realistic and meaningful trial endpoint. In most patients plexiform neurofibroma growth rate exceeded body growth rate. The youngest patients had the fastest plexiform neurofibroma growth rate, and clinical drug development should be directed toward this population. Age stratification for clinical trials for plexiform neurofibromas should be considered.

The involvement of hydroxyl free radicals in differentiation of the PC-12 rat pheochromocytoma cell line.

These experiments tested the differentiation properties of the PC-12 cell line under conditions of in vitro generation of OH&z.rad; free radicals by Fenton reaction. This involves the simultaneous addition of the following reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.025 mM), final concentrations. Superoxide dismutase activity, the increase of which is considered as a marker of differentiation, catalase and glutathione peroxidase enzyme activities were investigated, which all displayed significant increases after single and repeated interventions with hydroxyl free radicals, while the cell number remained nearly at the starting-value. It is known that the differentiation takes place when the cell number has reached a plateau. These data, therefore, suggest that hydroxyl free radicals can induce in vitro cell differentiation, and that they play a more complex role in cell physiology than simply causing oxidative damages. It is interesting that the cells can maintain high levels of these enzyme activities for a relatively long time (2 or 4 days) after a very short flux of hydroxyl free radicals.

On the useful role of OH&z.rad; free radicals in differentiation of cultured human fibroblasts.

The working hypothesis assuming that oxygen free radicals cannot be considered only as harmful by-products of the oxidative metabolism has been experimentally tested. Human fibroblasts were grown in culture from the following five types of tissues: (1) normal orbital fat; (2) orbital fat of patients with endocrine ophtalmopathy (EOP); (3) normal orbital muscle; (4) orbital muscle of EOP patients; (5) skin. These fibroblasts (second to 12th passages) were treated for 2x72 h with the Fenton reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.055 mM), final concentrations. This treatment caused a slowing down of the cell proliferation, induced various morphological signs of differentiation, and significantly increased (40-150%) the total superoxide dismutase (SOD) and catalase (CAT) activities of the fibroblasts. Authors suggest that the increased expression of these enzymes may play a general role in the cell differentiation mechanisms, meaning that the generation of oxygen free radicals is an essential, useful factor even during the early phases of development, and may not be taken only as a harmful process during aging.

Cytochemical studies on the fibroblast-preadipocyte relationships in cultured fibroblast cell lines.

The theoretical basis of adipogenesis has always been a matter of debate. One concept suggests that all types of adipocytes are derived from undifferentiated connective tissue cells, whereas another concept suggests that adipocytes develop from specialized cells only that are able to accumulate fat. Many conflicting data have been published with respect to the transition of fibroblasts into preadipocytes. For example, this transition has been declared as impossible for dermal and perimysial fibroblasts. The present study analysed spontaneous accumulation of fat in various types of fibroblasts from different origin (retroocular, skin, NIH/3T3, and L929). It was found that intense Oil Red O-positive triglyceride-containing droplets accumulated in practically all types of fibroblasts provided that the cells were cultured on glass surface. When the cells were cultured on plastic surfaces, lipid staining was inhibited in a variable manner: inhibition was virtually complete in skin fibroblasts, whereas in other types of fibroblasts, inhibition was only partial. It is concluded that all types of fibroblasts can accumulate fat spontaneously, and thus can be considered as preadipocytes. Therefore, interpretations of data obtained with cultures of fibroblasts with respect to adipogenesis have to be reconsidered.

Cell-mediated immunity in subjects immunized with HLA antigens.

Production of cellular immune reaction in subjects immunized against HLA antigens was studied by the capillary migration inhibition test in vitro and by the skin test in vivo. The majority of the immunized subjects (10 out of 13) developed a cell-mediated immunity to the donor antigens. Production of HLA antibodies was demonstrable in a smaller proportion of the cases. The complementary nature of humoral and cellular responsiveness was borne out by the present observations. The result of the skin test performed simultaneously with the migration inhibition test correlated well with the result in vitro.